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A Schiff Base-Derived Copper (II) Complex is a potent inducer of apoptosis in

colon cancer cells by activating the intrinsic pathway

Maryam Hajrezaie,1,2 Mohammadjavad Paydar,3 Soheil Zorofchian Moghadamtousi,1

Pouya Hassandarvish,1 Nura Suleiman Gwaram,4 Maryam Zahedifard,1,2 Elham

Rouhollahi,1 Hamed Karimian,1 Chung Yeng Looi,3 Hapipah Mohd Ali,4 Nazia Abdul

Majid,1 Mahmood Ameen Abdulla, 1

1 Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

2 Institute of Biological Science, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

3 Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

4 Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia

Maryam Hajrezaie email address: Maryam_hajrezaie@yahoo.com

Mohammadjavad Paydar email address: paydarmj@gmail.com

Soheil Zorofchian Moghadamtousi email address: soheil.zorofchian@gmail.com

Pouya Hassandarvish email address: pouya3132@gmail.com

Nura Suleiman Gwaram email address: Nura_suleiman@yahoo.com

Maryam Zahedifard email address: z_maryam81@yahoo.com

Elham Rouhollahi email address: e_rouhollahi@yahoo.com

Hamed Karimian email address: hamed_karimian61@yahoo.com

Chung Yeng Looi email address: looicy@um.edu.my

Hapipah Mohd Ali email address: hapipah@um.edu.my

Nazia Abdul Majid email address: nazia@um.edu.my

* Correspondence should be addressed to Mahmood Ameen Abdulla: ammeen@um.edu.my

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ABSTRACT

Metal-based drugs with extensive clinical applications hold great promise for the development of

cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have

attracted significant attention in the field of coordination chemistry and have become well known

for their extensive biological potential. In the present study, we examined the antiproliferative

effect of a Copper (II) complex derived from N,N’dimethyl ethylene diamine and a 2-

hydroxyacetophenone Schiff base ligand on HT-29 colon cancer cells. This study also evaluated

the induction of apoptosis by this compound and possible mechanisms of action. The

Cu(BrHAP)2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells,

with an IC50 value of 2.87 µg/ml after 72 h of treatment. HT-29 cells treated with Cu (II)

complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion

of the G1 cell population. At a concentration of 6.25 µg/ml, the Cu(BrHAP)2 compound caused

significant elevation in ROS production following perturbation of mitochondrial membrane

potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in

stained cells. Furthermore, the activation of caspases-3/7 and -9 was part of the Cu (II) complex-

induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis.

Meanwhile, there was no significant activation of caspase-8, possibly suggesting the lack of

involvement of the extrinsic pathway. Taken together, these results imply that the Cu(BrHAP)2

compound is a potential candidate for further in vivo and clinical colon cancer studies to develop

novel chemotherapeutic agents derived from metal-based agents.

Keywords: Schiff base compound; Colon cancer; HT-29 cells; Apoptosis; Cell cycle

1. Introduction

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Cancer is a debilitating disease that afflicts a substantial portion of the world population in all

generations and is a major health problem of global concern [1]. Among the various types of

cancer, colorectal cancer is the second and third most prevalent cancer among males and females

in the United States, respectively. In spite of all the considerable progress in protective methods

and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year

relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%,

respectively [2]. In addition, due to bitter controversy over optimal methods for early detection,

full compliance of patients with screening recommendations remains a major hindrance for

diagnosis at the early stages of cancer development. Development of resistance to chemotherapy

also represents a critical issue for which simultaneous treatment with various classes of

therapeutics to reduce the resistance has yielded some success [3]. Moreover, the numerous side

effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding and

immunosuppression, have made the process of treatment more complicated [4]. The highly

regulated programmed cell death process of apoptosis is a matter of great interest in oncology

and cancer therapy and represents a common molecular pathway for drug resistance and

carcinogenesis [5].

Maintenance of a constant cell number in the colonic mucosa is highly regulated through the

balance between apoptosis and cell proliferation. The perturbation in this balance leads to an

escape from normal cell number homeostasis and is associated with the progression of cancer

cells [6, 7]. Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells is

suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore,

apoptosis and the factors involved in its mechanism of action also present a window that can be

exploited for the improvement of potential therapeutic agents with high effectiveness and less

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adverse side effects [8]. Hence, screening for novel compounds capable of inducing apoptosis in

colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is

a significant need and represents a critical challenge in medicinal chemistry.

Metal complexes have been extensively utilized in clinics for centuries and have attracted

numerous inorganic chemists to analyze them, with the main focus being medical applications

[9, 10]. Copper, an essential trace element with an oxidative nature and bioessential activity in

human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of

copper in the body is evaluated in the form of complexes with organic compounds [11]. Schiff

bases are a critical class of compounds in medical chemistry that have demonstrated significant

chemotherapeutic and antibacterial application [12, 13]. Schiff base Cu(II) complexes revealed

great potential for antiproliferative, antibacterial and gastroprotective activity [14-18].This study

evaluated the anticancer potential of a copper (II) complex derived from N,N’dimethyl ethylene

diamine and 2-hydroxyacetophenone Schiff base ligand, Cu(BrHAP)2. Furthermore, the possible

apoptotic mechanism underlying this activity was also examined.

2. Materials and methods

2.1.Reagents and chemicals

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise

indicated. Stock solutions of tested compounds were prepared in dimethyl sulfoxide (DMSO)

and stored at -20°C, protected from the light.

2.2.Test material

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The previously described Copper (II) complex Cu(BrHAP)2 (Figure 1) was kindly supplied

by Prof. Dr. Hapipah Mohd Ali, Department of Chemistry, Faculty of Science, University of

Malaya, Kuala Lumpur, Malaysia [18].

Figure 1. Chemical structure of Cu(BrHAP)2

2.3.Cell culture and viability measurement

HT-29 human colon cancer cells and CCD 841 normal human colon epithelial cells were

purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained

routinely in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville,

MD) containing 10% fetal bovine serum, 100 µg/mL streptomycin and 100 U/mL penicillin G at

37°C in a humidified atmosphere of 5% CO2/95% air. The cells were plated at a fitting density in

tissue culture flasks (Corning, USA) according to each experimental scale. Cell viability was

measured by a conventional MTT [3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium

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bromide] reduction assay. After 48 h exposure to six concentrations of Cu(BrHAP)2, cells were

treated with MTT solution (2 mg/ml) for 2 h. The dark formazan crystals formed in intact cells

were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a

background using a microplate reader (Hidex, Turku, Finland). The IC50 value was determined

as the concentration of Cu(BrHAP)2 required to reduce the absorbance of treated cells to 50% of

the DMSO-treated control cells. All samples were prepared in triplicates.

2.4.LDH release assay

Measurement of lactate dehydrogenase (LDH) release is a biomarker for determining the

cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of

Cu(BrHAP)2 and Triton X-100 (positive control) for 48 h, and the supernatants of the untreated

and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 µl

of LDH reaction solution was added to each well, the plate was incubated at room temperature

for 30 min and the absorbance was read at 490 nm using a Tecan Infinite®200 Pro (Tecan,

Männedorf, Switzerland) microplate reader. The amount of formazan salt and intensity of red

color in treated and untreated samples were represented as the LDH activity of cells. The LDH

release level in cells treated with Cu(BrHAP)2 was expressed as a percentage of the positive

control.

2.5.Acridine orange/ Propidium iodide double staining

A propidium iodide (PI) and acridine orange (AO) double staining assay was carried out for

detection of apoptosis in the treated cells using a fluorescent microscope (Leica attached with Q-

Floro software) according to a standard procedure. HT-29 cells (5 × 104 cells/ml in a 25 ml

culture flask) were plated, treated with Cu(BrHAP)2 at the IC50 concentration and incubated for

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24, 48 and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed

under a UV-fluorescent microscope (Olympus BX51) within 30 min.

2.6.Cell cycle analysis

In brief, HT-29 cells (1 × 104 cells/well in 96-well plate) were supplemented with

Cu(BrHAP)2 (2 µg/ml) or DMSO (negative control) for 24 h. The live cells were then incubated

with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as

described by the manufacturer’s instructions, they were visualized and analyzed using the

Cellomics ArrayScan HCS reader (Thermo Scientific). The fluorescence intensities of the dyes

were measured using a target activation bioapplication module.

To confirm the result of the fluorescence cell cycle analysis, HT-29 cells (5×104 cells/ml)

were treated with Cu(BrHAP)2 for 24, 48 and 72 h for flow cytometry analysis. After incubation,

HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow

cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by

mixing them with 700 µl of cold ethanol (90%) and were then kept at 4°C overnight. Treated

HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the

cells in PBS, 25 µl of RNase A (10 mg/ml) and 50 µl of propidium iodide (PI) (1 mg/ml) were

added to the fixed cells for 1 h at 37 ˚C. The added RNase A limited the ability of PI to bind to

only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow

cytometer (BD FACSCanto TM II).

2.7.ORAC assay

The oxygen radical antioxidant capacity (ORAC) assay was carried out based on the

protocols described in detail previously [20]. In brief, Cu(BrHAP)2 at the concentration of 100

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µg/mL was used for this assay in a total reaction volume of 200 µL. The experiment was

performed in a black 96-well microplate with 25 µL of compound, blank (solvent/PBS), standard

(trolox) or positive control (quercetin). The plate was then supplemented with the working

fluorescein solution (150 µL), followed by a 5 min incubation at 37°. The total volume of 200

µL was made up by adding 25 µL of AAPH working solution. Fluorescence intensity was

measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2

min for 2 h. The result was quantified by calculating the differences of area under the

fluorescence decay curve (AUC) of samples and blank. The values were Trolox equivalents

(TE).

2.8.Measurement of reactive oxygen species generation (ROS)

In brief, HT-29 cells (1× 104 cells/mL) were seeded in 96-well plates and treated with

different concentrations of Cu(BrHAP)2 and DMSO (negative control) for 24 h. After 30 min

treatment with dihydroethidium (DHE) dye, cells were fixed and washed with wash buffer as

described by the manufacturer’s instructions. In the presence of superoxides, DHE dye is

oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at

an extension wavelength of 520 nm and an emission wavelength of 620 nm.

2.9. Multiple Cytotoxicity Assay

The critical factors for monitoring the cell health, namely, cell loss, changes in cell

permeability, cytochrome c release, mitochondrial membrane potential changes, nuclear size and

morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as

described in detail previously [19]. Plates with stained cells were analyzed using the ArrayScan

HCS system (Cellomics, PA, USA).

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2.10. Measurement of Caspase Activities

Caspase-3/7, -8 and -9 activities were determined using the commercial Caspase-Glo® 3/7, 8

and 9 assay kit (Promega, Madison, WI). HT-29 cells (1.0 × 104 cells/well) were seeded

overnight in white-walled 96-well plates and treated with different concentrations of

Cu(BrHAP)2 for 24 h. According to the manufacturer’s protocol, the treated cells were

supplemented with caspase-Glo reagent (100 µl) and incubated at room temperature for 30 min.

The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic

tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were

analyzed using a Tecan Infinite®200 Pro (Tecan, Männedorf, Switzerland) microplate reader.

2.11.Measurement of NF-κB activity

In brief, HT-29 cells (1.0 × 104 cells/well in a 96-well plate) were treated with different

concentrations of Cu(BrHAP)2 for 3 h, followed by stimulation with TNF-α (1 ng/ml) for 30

min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-

κB (NF-κB) activation kit (Thermo Scientific) according to the manufacturer's instructions. Next,

an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-κB

intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication

software. The average intensity of 200 cells/well was determined. The ratios for untreated,

treated and TNF-α-stimulated cells were compared.

2.12. Statistical Analysis

All the experiments were performed at least three times independently. The results were

presented as the mean ± standard deviation (SD) of the number of experiments shown in the

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legends. An analysis of variance (ANOVA) was carried out using the prism statistical package

(GraphPad Software, USA). P < 0.05 was considered statistically significant.

3. Results

3.1.The effect of Cu(BrHAP)2 on human normal and cancer cells of the colon

Initially, the cytotoxicity of Cu(BrHAP)2 was tested on HT-29 and CCD 841 cell lines. The

IC50 values of the Schiff base compound were determined based on the result collected from

three independent MTT experiments. As indicated in Table 1, Cu(BrHAP)2 elicited a significant

cytotoxicity and cell inhibitory effect after 24, 48 and 72 h of treatment on HT-29 cell.

Table 1. Inhibitory effects of Cu(BrHAP)2 on the proliferation of human normal and cancer

cells. Cells were treated with various concentrations of Cu(BrHAP)2 for 24, 48 and 72 h. The

IC50 values were analyzed by non-linear regression analysis.

Cell line Classification IC50 (µg/ml)

24 h 48 h 72 h

HT-29 Colon cancer cells 2.87 ± 0.21 1.93 ± 0.37 1.44 ± 0.26

CCD 841 Normal colon cells 50 ˂ 50 ˂ 50 ˂

3.2.Cu(BrHAP)2 -induced LDH release

Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of

membrane integrity, apoptosis or necrosis. The cytotoxicity of the Cu(BrHAP)2 compound, as

determined by the LDH release assay, was quantified on HT-29 cells treated with various

concentrations of the Schiff base compound for 48 h. Cu(BrHAP)2 induced a significant

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elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 µg/ml concentrations

compared to the control cells (Figure 2).

Figure 2. The LDH release assay revealed the significant cytotoxicity of the Cu(BrHAP)2

compound on HT-29 cells at the 6.25 and 12.5 µg/ml concentrations.

3.3.Quantification of apoptosis using phase-contrast microscopy and AO/PI double-

staining

Morphological changes in HT-29 cells treated with Cu(BrHAP)2 compound were observed

under a fluorescent microscope at 24, 48 and 72 h. The cells were scored under a fluorescent

microscope to analyze viable cells, early apoptosis and late apoptosis. Early apoptosis, defined as

intervening AO within the fragmented DNA, was observed under bright green fluorescence. At

the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48

h of treatment with Cu(BrHAP)2, moderate apoptosis was observed in the form of blebbing and

nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the

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presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after

72 h of treatment (Figure 3). The results showed that the Cu(BrHAP)2 compound induced

morphological features of apoptosis in a time-dependent manner.

Figure 3. (A) Untreated HT-29 cells after 72 h demonstrated normal structure without prominent

apoptosis or necrosis. Early apoptosis features, including blebbing and chromatin condensation,

were observed after (B) 24 and (C) 48 h of treatment with Cu(BrHAP)2. (D) Late apoptosis was

noticed after 72 h of Cu(BrHAP)2 treatment (magnification: 200×). VI: Viable cells; BL:

Blebbing of the cell membrane; CC: Chromatin condensation; LA: Late apoptosis.

3.4. Cu(BrHAP)2 -induced G1/G2 cell cycle arrest

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Cell cycle arrest of cells treated with 2 µg/ml Cu(BrHAP)2 was carried out using BrdU

and Phospho-Histone H3 dyes to determine whether HT-29 cells are arrested at the S/M phases.

Comparison of treated and untreated cells, as shown in Figure 4, demonstrated that there is no

cell cycle arrest in the S/M phases.

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Figure 4. (A) Effect of Cu(BrHAP)2 on cell cycle arrest in the S/M phase. After incubation with

DMSO or Cu(BrHAP)2 at 2 µg/ml for 24 h, HT-29 cells were collected, stained with BrdU and

Phospho-Histone H3 and subjected to the Cellomics ArrayScan HCS reader for cell cycle

analysis. (B) Representative bar charts indicating that Cu(BrHAP)2 treatment caused no

significant changes in BrdU or Phospho-Histone H3 fluorescence intensities in treated HT-29

cells. Cu(BrHAP)2 induced no cell cycle arrest at the S/M phases in treated HT-29 cells. Data

were expressed as the mean ± SD of fluorescence intensity readings for three independent

experiments.

The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the

G1/G2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell

cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M

phases. Meanwhile, significant cell cycle arrest in the G1 phase was observed for HT-29 cells

after 24 and 48 h of treatment (Figure 5).

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Figure 5. Effect of Cu(BrHAP)2 on cell cycle progression in HT-29 cells as assessed by flow

cytometry. After incubation with Cu(BrHAP)2 for 24 and 48 h, significant cell cycle arrest in the

G1 phase were observed.

3.5.ORAC anti-oxidant activity assay

Antioxidant capacity was measured by ORAC assay, which is the only assay that involves

the use of peroxyl radical as a pro-oxidant and quantifies activity via the area under the curve

(AUC) technique. In our experiment, quercetin was used as a positive control. The result

demonstrated that Cu(BrHAP)2 exhibited low to moderate antioxidant activity compared to

quercetin (Table 2).

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Table 2. Antioxidant capacity of the compound was determined by the ORAC method.

3.6.Effect of Cu(BrHAP)2 on Reactive Oxygen Species (ROS) formation

HT-29 cells were treated with different concentrations of Cu(BrHAP)2 for 24 h and stained

with DHE dye to determine the influence of the Schiff base compound on ROS production. The

fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence

microplate reader. As depicted in Figure 6, exposure to the Schiff base compound caused a

significant elevation in the ROS levels of treated HT-29 cells at the 6.25 µg/ml concentration.

Figure 6. Effect of Cu(BrHAP)2 on ROS production. After incubation with DMSO or

Cu(BrHAP)2 for 24 h, HT-29 cells were collected, stained with DHE and subjected to a

fluorescence microplate reader.

Compound net AUC mM of Trolox per 100μg/ml μM of Trolox per 100μg/ml

Cu(BrHAP)2 1765785 0.0056107 4.943

Quercetin 6353346 0.0214108 21.411

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3.7.Effects of Cu(BrHAP)2 on nuclear morphology, membrane permeability, mitochondrial

membrane potential (MMP) and cytochrome c release

To investigate the induction of apoptosis by Cu(BrHAP)2, nuclear morphological changes in

HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7, Hoechst

33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic

chromatin changes, emerged in some cells after treatment with Cu(BrHAP)2. Meanwhile, the

permeability of treated cells was also elevated. Mitochondria are the main source for the

production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and

survival of cells. The reduction in fluorescence intensity depicted in Figure 6 reflects the

significant decrease of MMP in the cells treated with the Schiff base compound. Meanwhile,

Cu(BrHAP)2 triggered the translocation of cytochrome c from mitochondria into the cytosol

during apoptosis in HT-29 cells.

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Figure 7. (A) Representative images of HT-29 cells treated with medium alone and 2 µg/ml of

Cu(BrHAP)2 and stained with Hoechst 33342 for nuclear, cytochrome c, membrane

permeability, and mitochondrial membrane potential dyes. Cu(BrHAP)2 induced a noteworthy

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elevation in membrane permeability and cytochrome c release and a marked reduction in

mitochondrial membrane potential (magnification: 200×). (B) Representative bar charts

indicating dose-dependent increased cell permeability, reduced MMP and increased cytochrome

c release in treated HT-29 cells.

3.8.Caspase activation

The elevation in ROS production associated with a collapse in MMP may lead to the

activation of the caspase cascade. To investigate caspase activation, the bioluminescent

intensities representing caspase-3/7, -8, -9 activities were quantified in HT-29 cells treated with

different concentrations of Cu(BrHAP)2 for 24 h. As shown in Figure 8, significant elevation in

the activity of caspase -3/7 at the 6.25 µg/ml concentration and caspase -9 at the 6.25 and 12.5

µg/ml concentrations was observed in Cu(BrHAP)2-treated cells, while no significant change in

the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the

apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via

the intrinsic pathway, but not the extrinsic pathway.

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Figure 8. Relative luminescence expression of Caspases 3/7, 8 and 9 in HT-29 cells treated with

various concentrations of Cu(BrHAP)2.

3.9.NF-κB Translocation

Nuclear factor kappa B (NF-κB) is a transcription factor that has a critical role in cytokine

gene expression. NF-κB activation and translocation to the nucleus to enable DNA-binding

activity and facilitate target gene expression is mediated by inflammatory cytokines such as

tumor necrosis factor-α (TNF-α). The Cu(BrHAP)2 Schiff base compound did not exhibit any

inhibitory effect on translocation of TNF-α-stimulated NF-κB in HT-29 treated cells, and TNF-

α-stimulation led to NF-κB translocation from the cytoplasm to the nucleus (Figure 9).

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Figure 9. (A) Photographs of the intracellular targets of stained HT-29 cells that were treated

with Cu(BrHAP)2 (2 µg/ml) for 3 h and then stimulated for 30 min with 1 ng/ml TNF-α (NF-κB

activation). (B) Representative bar chart indicating that Cu(BrHAP)2 treatment caused no

changes in TNF-α-induced NF-κB nuclear translocation in HT-29 cells.

4. Discussion

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Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a

reduction in apoptosis incidence serve as initial characterizations for its progression [21]. One of

the defense procedures in multicellular organisms is the destruction of undesirable cell

development, which is defined as programmed cell death. Apoptosis is the most noticed

programmed cell death mechanism and is characterized by distinct morphological changes such

as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane and

chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and

cell proliferation leads to cancer incidence [24], and agents that can induce apoptosis are known

to have potential anticancer effects [25, 26]. Apoptosis pathways are effective targets for cancer

therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to

regulate key events or molecules in apoptosis-inducing signal transduction pathways [27]. In the

present study, the Cu(BrHAP)2 Schiff base compound was evaluated for its ability to inhibit the

growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a

suitable model for colon cancer studies [28-30]. The Cu (II) complex used in this study exhibited

significant inhibition of HT-29 cell growth, with the IC50 values ranging from 2.87 at 24 h to

1.44 at 72 h. The results indicated that the Schiff base complex induced an antiproliferative

effect on human colon cancer cells in a time- and dose-dependent manner. Meanwhile, the non-

tumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the

compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the

level of LDH release from treated cells. Considerably elevated LDH release showed that the

cytotoxicity of the Cu(BrHAP)2 compound possibly occurred via the loss of membrane integrity,

whether through activation of apoptosis or the necrosis pathway [31].

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The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and

AO/PI double-staining following treatment of HT-29 cells with the compound included some

signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing and DNA fragmentation

[32, 33]. We found that the number of cells with early apoptosis features was higher at earlier

stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis

characterizations were dominant among treated HT-29 cells. Concurrent detection of late

apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis

may have progressed into necrosis due to the prolonged incubation with the Schiff base

compound.

To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II)

complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone

H3 staining along with flow cytometry [34-36]. BrdU dye can attach to the synthesized DNA of

replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells

in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double

staining assay indicated that there were no significant changes in the number of cells in the S/M

phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the

possibility that the cells were arrested in the G1 or G2 phase of the cell cycle. Thus, the flow

cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the

results demonstrated significant cell cycle arrest at G1 after 24 and 48 h of treatment, suggesting

proliferative suppression via induction of apoptosis [37, 38].

Perturbation of mitochondrial membrane potential is one of the earliest intracellular events

that occurs following the induction of apoptosis [39]. As the main source of cellular ROS and

adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling

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the survival or death of cells. After confirming that the Cu(BrHAP)2 Schiff base compound did

not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the

induction of ROS production in treated cells was analyzed. According to our study, after

exposing the Cu (II) compound to HT-29 cells and analyzing the levels of ROS, it was

demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a

compound concentration of 6.25 µg/ml.

In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis

through metal-induced ROS [40]. The intrinsic or mitochondrial-dependent signaling pathway

involves different factors of non-receptor-mediated stimuli that induce intracellular signals.

These signals, mainly through the p53 protein, act on the mitochondrial-initiated events.

Excessive ROS production is a negative signal that can result in the failure of suppression of

antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane

potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the

function of mitochondria in treated HT-29 cells. As shown in Figure 7, changes in MMP after

treatment with the Cu(BrHAP)2 Schiff base compound leading to the membrane depolarization

of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the

mitochondria matrix. The result implies that the induction of apoptosis by Cu (II) Schiff base

complexes may be associated with the mitochondrial pathway [26, 41, 42]. One of the important

signals to initiate the procedure of apoptosis is cytosolic cytochrome c. The release of

cytochrome c into the cytosol and reduction of its levels in the mitochondria have been shown to

occur as a result of changes in MMP [30]. As the result illustrated, the synthetic Schiff base

compound also led to an increase in the level of cytochrome c in the cytosol compared to the

control.

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The excessive production of ROS from mitochondria and the collapse of MMP may activate

the downstream caspase molecules and consequently lead to apoptotic cell death. After the

binding of cytochrome c to apoptotic activating factor-1, caspase-9 is activated via apoptosome

formation, which leads to active caspase-3/7, the most effective caspase with many cellular

targets [43]. In the extrinsic pathway, apoptosis is mediated by death receptors. As an example,

FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44]. Caspase-

8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46].

In our study, the Cu(BrHAP)2 schiff base compound induced significant elevation in the

caspase-3/7 and -9 activities compared to the control. Meanwhile, there was no activation of

caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic

mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway.

The supporting evidence of LDH release, ROS production, MMP suppression, elevation in

the level of cytochrome c and activation of caspase-3/7 and -9 demonstrated the promising

anticancer activity of the Cu(BrHAP)2 Schiff base compound against the HT-29 colon cancer

cell line via the intrinsic mitochondrial pathway.

Acknowledgments

The authors would like to thank the University of Malaya for supporting this project

through the PV069-2012A grant and the Ministry of Higher Education Malaysia for their support

through the HIR-MOHE (E0045-20001) grant.

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